Nuclear processes (i.e. transcription, replication, and repair) require the accessibility of specific DNA regulatory sequences to trans-acting factors. Current models of gene regulation mediated by changes in chromatin structure have been built upon the findings of many individual loci. This proposal aims to develop models for coordinating changes in higher-order chromatin structure for multiple genes over long distances. High throughput automation of an inter-nucleosomal cleavage-and-detection protocol developed in the lab will enable us to investigate long-range nucleosome positioning for multiple genes. Bioinformatics will be used to map nucleosome positions and may lead to the identification of novel regulatory sites. The HOX gene clusters, where differential gene expression is essential for hematopoietic differentiation, will be studied. The Polycomb group and trithorax group proteins are directly involved at the HOX gene clusters and biochemical and genetic assays will be used to examine how these chromatin remodelers regulate nucleosome positioning to mediate HOX gene expression. The proposed methodologies will allow us to better understand how complex genetic events can be regulated by changes in higher-order chromatin structure.